SIRT 1 activator including syringaresinol

ABSTRACT

The present invention relates to a compound of Formula 1, and an SIRT 1 activator including, as an active ingredient, derivatives thereof or pharmaceutically acceptable salts thereof. The present invention also relates to a composition including the SIRT 1 activator for detoxification, for the improvement of metabolic disorders, for the prevention or improvement of eye diseases, or the prevention or improvement of immune diseases.

TECHNICAL FIELD

The present disclosure relates to an SIRT1 activating agent containing acompound of Chemical Formula 1, a derivative thereof or apharmaceutically acceptable salt thereof as an active ingredient. Thepresent disclosure also relates to a composition for detoxification,improvement of metabolic disorder, prevention or improvement of eyedisease or prevention or improvement of immune disease containing theSIRT1 activating agent.

BACKGROUND ART

Smoking is known as an important risk factor of many diseases which arethe leading causes of human death. The major disease caused by smokinginclude lung cancer, chronic obstructive pulmonary disease (COPD),coronary artery disease, cerebrovascular disease such as stroke, heartfailure, circulatory disease, laryngopharyngeal cancer, oral cancer,etc.

Cigarette smoke contains over 4,000 toxic substances, includingcarcinogens. Among them, tar which is a mixture of several toxicsubstances and nicotine are known as important harmful ingredients.Since the harmful ingredients contained in tobacco suppress SIRT1 whichis known as human longevity gene, they render cigarette smokersvulnerable to inflammations or severe diseases such as chronicobstructive pulmonary disease (COPD). Also, smoking is known to causeabnormality in mitochondrial function.

Owing to the change in lifestyles with industrial development andsurplus high-calorie food, humankind is exposed to the risk of metabolicdiseases represented by obesity, type 2 diabetes, hyperlipidemia, fattyliver, etc. Considering that, among them, obesity is the major cause ofmetabolic diseases such as type 2 diabetes, hyperlipidemia and fattyliver, the other metabolic diseases could be prevented by preventing orimproving obesity.

Obesity is most commonly caused by energy input exceeding consumption ofenergy by the body. Specifically, surplus energy is stored in adiposetissue in the form of triglyceride. Although the adipose tissue canstore a large amount of surplus energy as if a rubber balloon, the sizeof adipocytes is increased at the same time. In addition, if the amountof surplus energy to be stored exceeds the capacity of the adiposetissue, lipid dysregulation lipotoxicity occur in which energy is storedin other tissues such as muscle, liver, etc. The fat stored in eachtissue is changed into free fatty acid through lipolysis. The free fattyacid is known to inhibit insulin signaling through signaling mechanismsinvolving JNK, PKC, etc. The inhibition of insulin signaling necessarilyinduces insulin resistance and, as a result, leads to various metabolicdiseases such as hyperglycemia, hyperlipidemia, hypercholesterolemia,type 2 diabetes, fatty liver, etc. As such, obesity is a severe problemsince it is not just an appearance concern but is accompanied by variousadult diseases.

The retina is an organ considered part of the central nervous system.Fully grown retinal cells do not normally divide like most neuronsexisting in the brain. Therefore, if the function of the retinal cellsis degenerated, disorder can occur easily in tissue or organ level whencompared with other system and, as a result, aging proceeds fast.Oxidative stress is a major cause of the degeneration of the retinalcell function because the retina, optic nerve, photoreceptor cells andlens constituting eyes are consistently exposed to the cause ofoxidative damage such as light and UV. Upon oxidative damage,mitochondrial DNA in intraocular cells is damaged. Since mitochondrialack enzymes that can repair the damaged DNA, the damaged mitochondrialDNA is accumulated in the cells with time. If the mitochondrial DNAbecomes unstable, modification occurs in mitochondria proteinsynthesized therefrom. As a result, the mitochondrial membrane potentialdecreases, followed by decreased production of mitochondrial energy(ATP) and relatively increased generation of reactive oxygen species.Consequently, modification occurs in the DNA, protein and lipidconstituting the cells and aging of eyes or eye disease such as maculardegeneration, uveitis, glaucoma, diabetic retinopathy and cataractoccurs. Accordingly, eye disease can be prevented or improved byinhibiting destabilization of mitochondrial DNA. However, the mostwidely known method of delaying eye aging at present is an indirectmethod of using antioxidant to reduce oxidative stress.

When the human body is invaded by viruses, bacteria, etc., the infectedcells produce histamine, kinin, etc., which dilate capillaries of thedamaged part and thus allow various immune cells including macrophagesand killer cells to easily reach the wound site. After the immune cellssuch as macrophages engulf the invading viruses, bacteria, etc., variousenzymes in lysosomes break them down and the immune response isterminated.

Immune response is an essential for maintenance of the body'shomeostasis along with energy metabolism. Innate immunity is a primarydefense mechanism against nonspecific sources of infection from outsideand defends our bodies from incessant invasion from outside throughinflammatory responses. The inflammatory responses are strictlyregulated because they have a great impact on cells and tissues. If theinflammatory response is not controlled normally, various immunediseases such as hay fever, rheumatoid arthritis, allergy, atopicdermatitis, etc. can occur. Moreover, since chronic inflammation whereinthe expression of inflammation-related genes is increased even in theabsence of external pathogens can cause metabolic diseases such as type2 diabetes by affecting insulin resistance, the regulation of immuneresponse is a prerequisite for the control of homeostasis ofindividuals.

DISCLOSURE Technical Problem

The present disclosure is directed to providing an SIRT1 activatingagent which activates SIRT1.

The present disclosure is also directed to providing a compositionhaving excellent detoxifying effect.

The present disclosure is also directed to providing a compositionhaving excellent effect of preventing or improving metabolic disorder,specifically having effect of preventing or improving metabolic disorderby regulating fat metabolism.

The present disclosure is also directed to providing a compositionhaving excellent effect of preventing or improving eye disease,specifically having effect of preventing or improving age-related eyedisease by increasing sirtuin 1 (SIRT1) expression and mitochondrialsynthesis in retinal cells.

The present disclosure is also directed to providing a compositionhaving excellent effect of preventing or improving immune disease,specifically having effect of preventing or improving immune disease bypromoting SIRT1 expression in immune cells and differentiation into type2 immune cells.

Technical Solution

In an aspect, the present disclosure provides an SIRT1 activating agentcontaining a compound of Chemical Formula 1, a derivative thereof or apharmaceutically acceptable salt thereof as an active ingredient:

wherein

R₁, R₂, R₃ or R₄ is independently an unbranched or branched C₁-C₁₈ alkylgroup, C₁-C₁₈ alkoxy group, C₁-C₁₈ alkenyl group, C₁-C₁₈ alkynyl groupor C₃-C₆ cyclic alkyl group, and

R₅, R₆, R₇, R₈, R₉, R₁₀ or R₁₁ is independently hydrogen or anunbranched or branched C₁-C₁₈ alkyl group, C₁-C₁₈ alkoxy group, C₁-C₁₈alkenyl group, C₁-C₁₈ alkynyl group or C₃-C₆ cyclic alkyl group.

In another aspect, the present disclosure provides a composition fordetoxification containing the SIRT1 activating agent.

In another aspect, the present disclosure provides a composition forpreventing or improving metabolic disorder containing the SIRT1activating agent.

In another aspect, the present disclosure provides a composition forpreventing or improving eye disease containing the SIRT1 activatingagent.

In another aspect, the present disclosure provides a composition forpreventing or improving immune disease containing the SIRT1 activatingagent.

Advantageous Effects

A sirtuin 1 (SIRT1) activating agent according to the present disclosureincreases SIRT1 expression and enhances SIRT1 activity.

A composition according to the present disclosure has superiordetoxifying effect, particularly superior effect of detoxifyingsmoking-induced toxicity by promoting SIRT1 activity and cellularactivity that have been decreased due to smoking.

The composition according to the present disclosure has excellent effectof preventing or improving metabolic disorder, specifically obesity,type 2 diabetes, hyperlipidemia or fatty liver, by increasing SIRT1expression, inhibiting fatty acid synthesis and promoting fatty acidoxidation at the same time, and increasing PGC-1 expression.

Also, the composition according to the present disclosure has excellenteffect of preventing or improving eye disease, specifically age-relatedeye disease, by promoting SIRT1 expression, enhancing mitochondrialbiosynthesis and restoring mitochondrial function in retinal cells.

The composition according to the present disclosure can enhance immunityby promoting SIRT1 expression and inhibiting inflammatory response inimmune cells and converting and immune cells to type 2 immune cells andparticularly has excellent effect of preventing or improving immunedisease such as allergy, atopic dermatitis, hay fever or rheumatoidarthritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically describes a method of isolating and purifyingsyringaresinol from a ginseng berry extract.

FIG. 2 shows the SIRT1 expression promoting effect of syringaresinol.

FIG. 3 shows the cellular activity promoting effect of syringaresinol.

FIG. 4 shows the change in expression of the SIRT1 gene in humanadipocytes, hepatocytes and myocytes treated with 20, 50 and 100 μMsyringaresinol.

FIG. 5 shows the decrease in expression of fatty acid synthesis-relatedgenes in human adipocytes treated with 50 μM syringaresinol.

FIG. 6 shows the decrease in expression of fatty acid synthesis-relatedgenes in human hepatocytes treated with 50 μM syringaresinol.

FIG. 7 shows the decrease in expression of synthesis-related genes inhuman myocytes treated with 50 μM syringaresinol.

FIG. 8 shows the increase in expression of fatty acid oxidation-relatedgenes in human adipocytes treated with 50 μM syringaresinol.

FIG. 9 shows the increase in expression of fatty acid oxidation-relatedgenes in human hepatocytes treated with 50 μM syringaresinol.

FIG. 10 shows the increase in expression of fatty acid oxidation-relatedgenes in human myocytes treated with 50 μM syringaresinol.

FIG. 11 shows the increase in fatty acid oxidation in human adipocytes,hepatocytes and myocytes treated with 50 μM syringaresinol.

FIG. 12 shows the increase in expression of the energy metabolismregulating gene PGC-1α in human adipocytes, hepatocytes and myocytestreated with 50 μM syringaresinol.

FIG. 13 shows the increase in expression of the energy metabolismregulating gene PGC-1β in human adipocytes, hepatocytes and myocytestreated with 50 μM syringaresinol.

FIG. 14 shows the increase in expression of the SIRT1 gene in agedretinal epithelial cells treated with syringaresinol.

FIG. 15 shows the mitochondrial biosynthesis level in aged retinalepithelial cells treated with syringaresinol.

FIG. 16 shows the energy (ATP) and reactive oxygen species production inaged retinal epithelial cells treated with syringaresinol.

FIG. 17 shows the increase in expression of the SIRT1 gene in humanperipheral blood mononuclear cells treated with syringaresinol.

FIG. 18 shows the inhibited reactive oxygen species production in humanperipheral blood mononuclear cells treated with syringaresinol.

FIG. 19 shows the decrease in expression of inflammatoryresponse-related genes in human peripheral blood mononuclear cellstreated with syringaresinol.

FIG. 20 shows the suppressed migration and tissue deposition of humanperipheral blood mononuclear cells treated with syringaresinol.

FIG. 21 shows the increase in expression of genes related withconversion to type 2 immune cells in human peripheral blood mononuclearcells treated with syringaresinol.

BEST MODE FOR CARRYING OUT INVENTION

In the present disclosure, the term “extract” is used as a broad conceptand refers to any substance extracted from a natural product, regardlessof extraction method, extraction solvent, extracted ingredients or thetype of extract.

As used herein, the term “derivative” refers to any compound havingsubstituent(s) at substitutable position(s) of the correspondingcompound. The substituent is not particularly limited. For example, thesubstituent may independently be a C₁₋₁₀ acyclic hydrocarbon group whichmay be substituted with hydroxyl, phenoxy, thienyl, furyl, pyridyl,cyclohexyl, alkylalcohol, alkyldialcohol or substituted phenyl; a C₅₋₆cyclic hydrocarbon group which may be substituted with hydroxyl,hydroxymethyl, methyl or amino; or a sugar residue, although not beinglimited thereto. As used herein, the term “sugar residue” refers to thegroup available on elimination of one hydrogen atom from a carbohydratemolecule. As such, it may mean, for example, a residue derived from amonosaccharide or an oligosaccharide.

As used herein, the term “pharmaceutically acceptable” means beingdevoid of substantial toxic effects when used with a usual medicinaldosage and thereby being approvable or approved by a regulatory agencyof the government or being listed in the US Pharmacopoeia or othergenerally recognized pharmacopoeia for use in animals, more particularlyin human.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt of the compound of the present disclosure which is pharmaceuticallyacceptable and possesses the desired pharmacological activity of theparent compound. The salt may include: (1) an acid addition salt formedwith an inorganic acid such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, etc. or formed with anorganic acid such as acetic acid, propionic acid, hexanoic acid,cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, ethane-1,2-disulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, 4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonicacid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylaceticacid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid or muconic acid; or(2) a salt formed when an acidic proton present in the parent compoundis replaced. In addition to the pharmaceutically acceptable salt, thecompound according to the present disclosure may include any salt,hydrate or solvate that can be prepared according to commonly employedmethods.

Hereinafter, the present disclosure is described in detail.

In an aspect, the present disclosure provides an SIRT1 activating agentcontaining a compound of Chemical Formula 1, a derivative thereof or apharmaceutically acceptable salt thereof as an active ingredient:

wherein

R₁, R₂, R₃ or R₄ is independently an unbranched or branched C₁-C₁₈ alkylgroup, C₁-C₁₈ alkoxy group, C₁-C₁₈ alkenyl group, C₁-C₁₈ alkynyl groupor C₃-C₆ cyclic alkyl group, and

R₅, R₆, R₇, R₈, R₉, R₁₀ or R₁₁ is independently hydrogen or anunbranched or branched C₁-C₁₈ alkyl group, C₁-C₁₈ alkoxy group, C₁-C₁₈alkenyl group, C₁-C₁₈ alkynyl group or C₃-C₆ cyclic alkyl group.

In an exemplary embodiment of the present disclosure, the compound maybe syringaresinol.

As used herein, the term “syringaresinol” refers to a lignan-basedcompound having a chemical structure represented by Chemical Formula 2.It may be synthesized chemically or extracted from one or more of flaxseed, phellodendri cortex, acanthopanacis cortex, sesame seed andginseng berry. The flax seed, phellodendri cortex, acanthopanacis cortexand sesame seed respectively include all parts of the plant, forexample, leaves, stem, root, fruit or seed and the ginseng berryincludes the rind or pulp of ginseng berry.

In the present disclosure, the “syringaresinol” may be obtained byextracting one or more of flax seed, phellodendri cortex, acanthopanaciscortex, sesame seed and ginseng berry with water, an organic solvent ora mixture of water and an organic solvent. The organic solvent includesone or more selected from a group consisting of alcohol, acetone, ether,ethyl acetate, diethyl ether, methyl ethyl ketone and chloroform,although not being limited thereto. The alcohol includes a C₁-C₅ loweralcohol and the C₁-C₅ lower alcohol includes one or more selected from agroup consisting of methanol, ethanol, isopropyl alcohol, n-propylalcohol, n-butanol and isobutanol, although not being limited thereto.

The SIRT1 activating agent according to the present disclosure maycontain 0.0001-10 wt % of the active ingredient based on the totalweight of the activating agent. However, the content of the activeingredient may be greater or smaller than the above range as long asSIRT1 activating effect is achieved and toxicity does not occur. Theabove-described range is appropriate not only to derive the effectdesired by the present disclosure and satisfy both the stability andsafety of the composition but also in terms of cost effectiveness.Specifically, if the content of the compound of Chemical Formula 1,specifically syringaresinol, is less than 0.0001 wt %, SIRT1 may not beactivated. And, if it exceeds 10 wt %, the safety and stability of theSIRT1 activating agent may be unsatisfactory. More specifically, theactive ingredient of the present disclosure may be contained in anamount of 0.0005-8 wt %, 0.001-6 wt % or 0.01-4 wt %.

The syringaresinol may be contained in an extract of flax seed,phellodendri cortex, acanthopanacis cortex, sesame seed or ginsengberry.

In an exemplary embodiment of the present disclosure, syringaresinol maybe isolated and purified from ginseng berry by a procedure including:preparing an alcohol extract of ginseng berry pulp; eluting the preparedalcohol extract with a solvent including one or more of water andalcohol and obtaining fractions thereof; and performing chromatography,specifically thin-layer chromatography (TLC), on the obtained fractionsusing an organic solvent as an eluent. The organic solvent may includeone or more selected from a group consisting of alcohol, acetone, ether,ethyl acetate, diethyl ether, methyl ethyl ketone and chloroform, andthe alcohol may include a C₁-C₅ alcohol. In an exemplary embodiment ofthe present disclosure, the composition may contain the syringaresinolpurified as described above as an active ingredient.

In another aspect, the present disclosure provides composition fordetoxification containing the SIRT1 activating agent.

As used herein, the term “detoxification” refers to removal of theaction of a toxic substance included in the body.

The SIRT1 activating agent can detoxify a toxic substance included inthe body, and specifically can detoxify smoking-induced toxicity.

Sirtuin 1 (SIRT1), which is one of deacetylases, deacetylates PGC-1α byphysically interacting with PGC-1α at several lysine sites, therebyactivating PGC-1α. The activated PGC-1α promotes mitochondrialbiosynthesis and removes cytotoxic substances such as reactive oxygenspecies by reacting with transcription factors. Accordingly, it isexpected to that smoking-induced toxicity can be detoxified by restoringSIRT1 activity that has been decreased due to smoking. The compound ofChemical Formula 1, specifically syringaresinol, can detoxifysmoking-induced toxicity by promoting SIRT1 activity and cellularactivity that have been decreased due to smoking. Accordingly, thecomposition containing the compound of Chemical Formula 1, specificallysyringaresinol, may have detoxifying effect.

In an exemplary embodiment of the present disclosure, the syringaresinolmay be contained in the composition as an extract of flax seed,phellodendri cortex, acanthopanacis cortex, sesame seed or ginsengberry. Specifically, it may be contained in a fraction which isparticularly effective for detoxification.

The composition for detoxification according to the present disclosuremay contain 0.001-20 wt %, specifically 0.01-10 wt %, more specifically0.1-5 wt % of the SIRT1 activating agent based on the total weight ofthe composition. This range is appropriate not only to derive the effectdesired by the present disclosure and satisfy both the stability andsafety of the composition but also in terms of cost effectiveness.Specifically, if the content of the SIRT1 activating agent is less than0.01 wt %, sufficient skin detoxifying effect may not be achieved. And,if it exceeds 20 wt %, the safety and stability of the composition maybe unsatisfactory.

In another aspect, the present disclosure provides a composition forpreventing or improving metabolic disorder, containing the SIRT1activating agent.

If fat metabolism can be regulated, obesity may be prevented andimproved since excessive fat accumulation can be prevented. One of themethods for preventing excessive fat accumulation is to control diet,i.e. calorie restriction. Through calorie restriction, body weight canbe decreased and metabolic disease can be improved by lowering bloodlipid concentration. It is known that calorie restriction leads toincreased SIRT1 expression and a similar phenomenon occurs as in calorierestriction when the activation of SIRT1 is induced. SIRT1 is anNAD⁺-dependent histone deacetylase which regulates energy metabolismsuch as glucose synthesis, fatty acid oxidation, etc. Accordingly, asubstance which promotes the expression of SIRT1 is expected to becapable of regulating fat metabolism and preventing or improvingmetabolic disorder such as obesity, type 2 diabetes, hyperlipidemia andfatty liver.

To sustain life, living organisms intake nutrients and digest, absorband store them as a source of energy. Also, they carry out variousbiological activities using energy released therefrom. This phenomenonis called “metabolism”.

As used herein, the term “metabolic disorder” refers to a condition inwhich a living organism including human or an animal cannot normallycarry out metabolic activities and includes carbohydrate metabolicdisorder or lipid metabolic disorder. The carbohydrate metabolicdisorder or the lipid metabolic disorder includes symptoms or diseasescaused by disorder of carbohydrate or lipid metabolism, specificallysymptoms or diseases caused by excessive fat accumulation induced bydisorder of fatty acid metabolism. Examples may include obesity, type 2diabetes, hyperlipidemia or fatty liver.

Syringaresinol can suppress fat accumulation in the body by increasingSIRT1 expression, inhibiting fatty acid synthesis and promoting fattyacid oxidation. This can be confirmed by the fact that syringaresinolsuppresses the expression of fatty acid synthesis-related genes such asADD1/SREBP1c, ACC and FAS, promotes the expression of fatty acidoxidation-related genes such as ACO, CPT1 and mCAD and enhances fattyacid oxidation. Also, syringaresinol can enhance consumption of body fatand suppress accumulation of surplus fat by promoting energy metabolism,specifically fat metabolism. This can be confirmed by the fact thatsyringaresinol increases the expression of peroxisomeproliferator-activated receptor coactivator 1 (PGC-1) which regulatesthe expression of energy metabolism-related genes. Accordingly, thecomposition containing syringaresinol may prevent or improve metabolicdisorder such as obesity, type 2 diabetes, hyperlipidemia or fattyliver, which his caused by carbohydrate or lipid metabolic disorder.

In an exemplary embodiment of the present disclosure, the syringaresinolmay be contained in the composition as an extract of flax seed,phellodendri cortex, acanthopanacis cortex, sesame seed or ginsengberry. Specifically, it may be contained in a fraction which isparticularly effective for improving metabolic disorder.

The composition according to the present disclosure may contain 0.001-20wt %, specifically 0.01-10 wt %, more specifically 0.1-5 wt % of theSIRT1 activating agent based on the total weight of the composition.This range is appropriate not only to derive the effect desired by thepresent disclosure and satisfy both the stability and safety of thecomposition but also in terms of cost effectiveness. Specifically, ifthe content of the SIRT1 activating agent is less than 0.01 wt %,sufficient effect of improving metabolic disorder may not be achieved.And, if it exceeds 20 wt %, the safety and stability of the compositionmay be unsatisfactory.

In another aspect, the present disclosure provides a composition forpreventing or improving eye disease, containing the SIRT1 activatingagent.

SIRT1, which is an NAD⁺-dependent histone deacetylase, is known torestore mitochondrial function and thus prevent cellular aging byincreasing mitochondrial biosynthesis in muscle, etc., and therebyincreasing the proportion of mitochondria having stable DNAs in cells.In particular, it is known that a transgenic mouse model of retinaldegeneration exhibits expression of SIRT1 at abnormal locations inretinal cells such as retinal ganglion cells, intraretina cells,photoreceptor cells and retinal pigment epithelial cells and alsoexhibits accelerated cell death. Accordingly, a substance that increasesSIRT1 expression will be able to prevent and improve eye disease,particularly age-related eye disease, by promoting mitochondrialbiosynthesis and restoring mitochondrial function in retinal cells andreducing instability of mitochondrial DNA, thereby reducing cell damageand death.

The compound of Chemical Formula 1, specifically syringaresinol, caninduce increased energy production and decrease of reactive oxygenspecies by increasing SIRT1 expression in aged retinal cells to a levelcomparable to that of young retinal cells, promoting mitochondrialsynthesis in retinal cells and recovering mitochondrial function.Accordingly, the composition containing the compound of Chemical Formula1, specifically syringaresinol, as an active ingredient can prevent orimprove eye disease by preventing or improving the aging of retinalcells.

As used herein, the term “eye disease” refers to a disease related witheyes and includes the disease of eyeball. And, as used herein, the term“age-related eye disease” includes not only the eye disease caused bythe decline in biological function with age but also the eye diseaseexhibiting symptoms similar to those occurring in the elderly whosebiological function is degenerated as compared to young people. In anexemplary embodiment of the present disclosure, the eye disease includesone caused by the disorder of mitochondrial function in retinal cells oroxidative stress. In another exemplary embodiment of the presentdisclosure, the eye disease includes age-related eye disease andincludes macular degeneration including age-related maculardegeneration, uveitis, glaucoma, diabetic retinopathy or cataract, butis not limited thereto.

In another exemplary embodiment of the present disclosure, thesyringaresinol may be contained in the composition as an extract of flaxseed, phellodendri cortex, acanthopanacis cortex, sesame seed or ginsengberry. Specifically, it may be contained in a fraction which isparticularly effective for prevention or improvement of eye disease.

The composition according to the present disclosure may contain 0.001-20wt %, specifically 0.01-10 wt %, more specifically 0.1-5 wt % of theSIRT1 activating agent based on the total weight of the composition.This range is appropriate not only to derive the effect desired by thepresent disclosure and satisfy both the stability and safety of thecomposition but also in terms of cost effectiveness. Specifically, ifthe content of the SIRT1 activating agent is less than 0.01 wt %,sufficient effect of improving eye disease may not be achieved. And, ifit exceeds 20 wt %, the safety and stability of the composition may beunsatisfactory.

In another aspect, the present disclosure provides a composition forpreventing or improving immune disease, containing the SIRT1 activatingagent.

Immune cells can be largely divided into type 1 immune cells whichdefend against pathogens by inducing pro-inflammatory responses and type2 immune cells which inhibit anti-inflammatory responses and help tissuereconstruction. It is known that the number of type 1 immune cells isgreatly increased in chronic inflammation and the chronic inflammationstate can be relieved by inducing differentiation of the type 1 immunecells into type 2 immune cells. This suggests that immune disease suchas allergy, atopic dermatitis, hay fever, rheumatoid arthritis, etc. canbe prevented and treated by controlling the type of immune cells.

It is known that activation of SIRT1 which is an NAD⁺-dependent histonedeacetylase that regulates various cellular responses including energymetabolism, cell cycle, DNA restoration, etc. leads to suppression ofchronic inflammation. Accordingly, a substance that promotes SIRT1expression will be able to improve immune disease by suppressinginflammatory response.

The compound of Chemical Formula 1, specifically syringaresinol,promotes SIRT1 expression in peripheral blood mononuclear cells (PBMCs)including immune cells such as B cells, T cells, macrophages, dendriticcells, NK cells, etc., suppresses inflammatory response as one of innateimmune responses and inhibits the production of reactive oxygen specieswhich are signaling mediators and also products of inflammatoryresponse. Also, since the compound of Chemical Formula 1, specificallysyringaresinol, promotes the expression of transcriptional regulatorsnecessary for inducement of differentiation into type 2 immune cells,such as PGC-1α and PGC-1β, and increases the expression of the genesthat are mainly expressed in type 2 immune cells, such as IL-10 andarginase I, the compound of Chemical Formula 1, specificallysyringaresinol, can promote the conversion of immune cells to type 2immune cells. Accordingly, the composition containing the compound ofChemical Formula 1, specifically syringaresinol, as an active ingredientcan enhance immunity and can prevent or improve immune disease,particularly chronic immune disease.

In an exemplary embodiment of the present disclosure, the immune diseaserefers to a disease related with a defensive mechanism against externalpathogens that may harm the body and means a disease exhibiting variousimmune responses such as antigen-antibody reaction, vasodilation, feverand inflammatory response. In another exemplary embodiment of thepresent disclosure, the immune disease includes one or more of allergy,atopic dermatitis, hay fever and rheumatoid arthritis, but is notlimited thereto.

In another exemplary embodiment of the present disclosure, thesyringaresinol may be contained in the composition as an extract of flaxseed, phellodendri cortex, acanthopanacis cortex, sesame seed or ginsengberry. Specifically, it may be contained in a fraction which isparticularly effective for prevention or improvement of immune disease.

The composition according to the present disclosure may contain 0.001-20wt %, specifically 0.01-10 wt %, more specifically 0.1-5 wt % of theSIRT1 activating agent based on the total weight of the composition.This range is appropriate not only to derive the effect desired by thepresent disclosure and satisfy both the stability and safety of thecomposition but also in terms of cost effectiveness. Specifically, ifthe content of the SIRT1 activating agent is less than 0.01 wt %,sufficient effect of preventing or improving immune disease may not beachieved. And, if it exceeds 20 wt %, the safety and stability of thecomposition may be unsatisfactory.

In an exemplary embodiment of the present disclosure, the compositionfor detoxification, preventing or improving metabolic disorder,preventing or improving eye disease or preventing or improving immunedisease may be a composition for oral administration.

In another exemplary embodiment of the present disclosure, thecomposition for detoxification, preventing or improving metabolicdisorder, preventing or improving eye disease or preventing or improvingimmune disease may be a food composition. The food composition includesan indulgence food or health food composition.

The composition may prevent or improve metabolic disorder such asobesity, type 2 diabetes, hyperlipidemia or fatty liver.

The food composition may prevent or improve eye disease such as maculardegeneration, uveitis, glaucoma, diabetic retinopathy or cataract.

The food composition may prevent or improve immune disease such asallergy, atopic dermatitis, hay fever or rheumatoid arthritis.

The formulation of the food composition is not particularly limited. Forexample, it may be formulated into tablet, granule, powder, liquid suchas drink, caramel, gel, bar, etc. Those skilled in the art may selectand add the ingredients commonly used in the art to each formulation ofthe food composition without difficulty. In this case, a synergic effectmay be achieved.

Determination of the dosage of the active ingredient is in the level ofthose skilled in the art. A daily dosage may be, for example, 0.1-5000mg/kg/day, more specifically 50-500 mg/kg/day. However, the dosage mayvary depending on various factors including the age and physicalcondition of a subject, the presence or absence of complication(s), orthe like, without being limited thereto.

In another exemplary embodiment of the present disclosure, thecomposition for detoxification, preventing or improving metabolicdisorder, preventing or improving eye disease or preventing or improvingimmune disease may be a pharmaceutical composition.

The pharmaceutical composition may exhibit detoxifying effect andspecifically may detoxify smoking-induced toxicity.

The pharmaceutical composition may prevent or improve metabolic disorderand specifically may prevent or improve obesity, type 2 diabetes,hyperlipidemia or fatty liver.

The pharmaceutical composition may prevent or treat eye disease andspecifically may prevent or treat macular degeneration, uveitis,glaucoma, diabetic retinopathy or cataract.

The pharmaceutical composition may enhance immunity and may prevent ortreat immune disease. Specifically, it may prevent or treat allergy,atopic dermatitis, hay fever or rheumatoid arthritis.

In an exemplary embodiment of the present disclosure, the pharmaceuticalcomposition may be administered orally or parenterally, e.g., rectally,topically, transdermally, intravenously, intramuscularly,intraperitoneally, subcutaneously, etc.

A formulation for oral administration may be tablet, pill, soft or hardcapsule, granule, powder, fine granule, liquid, emulsion or pellet,although not being limited thereto. These formulations may furthercontain, in addition to the active ingredient, a diluent (e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose or glycine), alubricant (e.g., silica, talc, stearic acid or polyethylene glycol) or abinder (e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methyl cellulose, sodium carboxymethyl cellulose orpolyvinylpyrrolidone). In some cases, they may further contain apharmaceutical additive such as a disintegrant, an absorbent, acolorant, a flavoring agent, a sweetener, etc. The tablet may beprepared according to the common mixing, granulation or coating method.

A formulation for parenteral administration may be injection, drop,lotion, ointment, gel, cream, suspension, emulsion, suppository, patchor spray, although not being limited thereto.

The dosage of the active ingredient of the pharmaceutical compositionaccording to the present disclosure will vary depending on the age, sexand body weight of a subject, particular pathological condition andseverity thereof, administration route or the discretion of a diagnoser.Determination of the dosage considering these factors is in the level ofthose skilled in the art. A daily dosage may be, for example, 0.1-100mg/kg/day, more specifically 5-50 mg/kg/day, although not being limitedthereto.

In another exemplary embodiment of the present disclosure, thecomposition for detoxification, preventing or improving metabolicdisorder, preventing or improving eye disease or preventing or improvingimmune disease may be a cosmetic composition.

The cosmetic composition may exhibit detoxifying effect and specificallymay detoxify smoking-induced toxicity.

The cosmetic composition may prevent or improve metabolic disorder andspecifically may prevent or improve obesity, type 2 diabetes,hyperlipidemia or fatty liver.

The cosmetic composition may enhance immunity and may prevent or treatimmune disease. Specifically, it may prevent or treat allergy, atopicdermatitis, hay fever or rheumatoid arthritis.

The cosmetic composition according to the present disclosure may beprovided in the form of any formulation suitable for topicalapplication. For example, it may be provided in the form of solution,oil-in-water emulsion, water-in-oil emulsion, suspension, solid, gel,powder, paste, foam or aerosol. These formulations may be preparedaccording to methods commonly employed in the art.

The cosmetic composition according to the present disclosure may furthercontain a humectant, an emollient, a surfactant, a UV absorbent, apreservative, a sterilizer, an antioxidant, a pH adjusting agent anorganic or inorganic pigment, a fragrance, a cooling agent or adeodorant. The amount of these ingredients may be determined easily bythose skilled in the art within a range not negatively affecting thepurpose and effect of the present disclosure. They may be added in anamount of 0.01-5 wt %, specifically 0.01-3 wt %, based on the totalweight of the composition.

MODE FOR INVENTION

Hereinafter, the present disclosure will be described in detail throughan example and test examples. However, the following example and testexamples are for illustrative purposes only and it will be apparent tothose of ordinary skill in the art that the scope of the presentdisclosure is not limited by the example and test examples.

[Example] Isolation and Analysis of Syringaresinol

1. Pretreatment of Ginseng Berry

Live ginseng berry was harvested. After removing the seed and rind ofthe ginseng berry, only the pulp was dried under sunlight or using hotair to obtain dried ginseng berry pulp.

2. Isolation of Syringaresinol from Ginseng Berry Pulp Extract andAnalysis Thereof

3 L of water or alcohol was added to 1 kg of the dried ginseng berrypulp. After extracting at room temperature or under reflux, followed byfiltering and concentration at 40-45° C. under reduced pressure, 300 gof a ginseng berry pulp extract was obtained. The extract was treatedwith ether to remove oil-soluble components and then crude saponin wasextracted with butanol and concentrated. Then, syringaresinol wasisolated and purified therefrom as follows. First, 194 g of the samplewas purified by reversed-phase (ODS) column chromatography. As theeluent, 100% water was used in the beginning. Subsequently, methanol wasincreased gradually by 10% and, finally, 100% methanol was used as theeluent. As a result, fractions GB-1 through GB-10 were obtained. Amongthe fractions, the fraction GB-3 which exhibits SIRT1 expressionactivity was concentrated and subjected to Sephadex LH-20 columnchromatography using 50% aqueous methanol. Among the resultingfractions, the fraction GB-3-6 exhibiting SIRT1 expression activity wasconcentrated and subjected to preparative silica gel TLC usingchloroform:methanol (10:1) as an eluent. As a result, an active fractionwith an R_(f) value of 0.67 was purified. This procedure isschematically described in FIG. 1.

Through NMR spectroscopic analysis and database search, the isolated andpurified active compound was identified as syringaresinol. Mass analysiswas conducted to confirm this. As a result of ESI-mass analysis in thepositive mode, [M+Na]⁺ (m/z=440.9) and [2M+Na]⁺ (m/z=858.9) peaks wereobserved and the molecular weight was calculated as 418. And, the resultof NMR spectroscopic analysis was as in Chemical Formula 3. Accordingly,the isolated and purified active compound was confirmed to besyringaresinol.

As such, syringaresinol was isolated from the ginseng berry pulp.

[Test Example 1] Evaluation of SIRT1 Expression Recovering Effect inGingival Fibroblasts

Experiment was conducted to evaluate whether syringaresinol can recoverSIRT1 expression that has been decreased due to smoking in gingivalfibroblasts.

1. Culturing of Gingival Fibroblasts

Gingival fibroblasts were obtained from tissue biopsies of patientshaving healthy gingiva. The tissue biopsy was put in a 15-mL test tubeholding Dulbecco's modified Eagle's medium (DMEM, Gibco Co., USA) andblood and impurities were removed by washing 3 times. Then, the tissuebiopsy was transferred onto a 100-mm tissue culture dish holding DMEMcontaining 10% fetal bovine serum (Gibco Co., USA) and 1% antibiotics(penicillin G 10,000 units/mL, amphotericin B 25 μg/mL, Gibco Co., USA)and finely cut to 1 mm² using a No. 15 blade. Then, 5-6 pieces wereuniformly distributed on a 60-mm culture dish. After incubation forabout 30 minutes in a 5% CO₂ incubator at 37° C. and 100% humidity suchthat the tissue was uniformly attached on the bottom of the culturedish, 3 mL of DMEM containing 10% fetal bovine serum and 1% antibioticswas added. The culture medium was exchanged with 2-3 day intervals untila single layer of cells was formed. Then, after removing the culturemedium and washing 2 times, the cells attached to the culture dish wereseparated using 0.25% trypsin/EDTA (1×, Gibco Co., USA) and transferredto a 100-mm tissue culture dish. The culture medium was exchanged with2-3 day intervals until sufficient cell proliferation was achieved andsubculturing was performed 5-6 times at a ratio of 1:3-4.

2. Preparation of Cigarette Smoke Condensate (CSC)

An automatic smoking machine (Heinr Borgwaldt RM 20, Germany) was usedto burn 40 3R4F cigarettes under the ISO smoking condition (puff volume:35 mL, puff duration: 2 seconds, puff frequency: 1 minute, butt length:tip paper +3 mm) and cigarette smoke condensate was collected using theCambridge glass fiber filter. The collected cigarette smoke condensatewas extracted in dichloromethane by sonicating for 30 minutes and thenconcentrated. The concentrated cigarette smoke condensate was dissolvedagain in dimethyl sulfoxide (DMSO) to a final concentration of 100mg/mL, filtered using a 0.22-μm syringe filter and kept at −70° C. untiluse.

3. PCR

The gingival fibroblasts subcultured 5-6 times were seeded onto a24-well plate, with 1×10⁴ cells per well. After culturing for 1 day, thecigarette smoke condensate was added with a concentration of 100 μg/mLand the syringaresinol obtained in Example was added after dissolving inDMSO to a concentration of 50 or 100 μg/mL. A control group was treatedwith DMSO corresponding to 1/1000 of the volume of the culture medium.Also, a group which was treated with neither the cigarette smokecondensate nor the test substance was prepared. After culturing for 2days, the cells were washed 2 times with cold PBS and RNA was extractedtherefrom using the TRIzol reagent (Invitrogen). Then, cDNA wassynthesized from the extracted RNA (1 μg/μL) using a reversetranscription system (Promega). The expression pattern of the SIRT1 andGAPDH genes was monitored using the synthesized cDNA and predesignedprimers and probes (Applied Biosystems; SIRT1, Hs01009006_m1; GAPDH,Hs99999905_m1). PCR reaction and analysis were carried out using theRotor-Gene 3000 system (Corbett Research, Sydney, Australia). The resultis shown in FIG. 2.

As can be seen from FIG. 2, syringaresinol increased SIRT1 expression ingingival fibroblasts, which had been decreased due to smoking, in aconcentration-dependent manner. Accordingly, it can be seen thatsyringaresinol can detoxify smoking-induced toxicity by increasing SIRT1expression.

[Test Example 2] Evaluation of Cellular Activity Promoting Effect inGingival Fibroblasts (MTT Assay)

Gingival fibroblasts subcultured for 5-6 times in substantially the samemanner as in Test Example 1 were seeded onto a 24-well plate, with 1×10⁴cells per well. After culturing for 1 day, cigarette smoke condensatewas added with a concentration of 100 μg/mL in substantially the samemanner as in Test Example 1 and the syringaresinol obtained in Examplewas added after dissolving in DMSO to a concentration of 50 or 100μg/mL. A control group was treated with DMSO corresponding to 1/1000 ofthe volume of the culture medium. Also, a group which was treated withneither the cigarette smoke condensate nor the test substance wasprepared. After culturing for 2 days and adding 300 μL of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;Sigma, USA) dissolved in physiological saline per each well, the cellswere cultured for 4 hours. After removing the culture medium anddissolving formazan crystals by adding 200 μL of dimethyl sulfoxide(DMSO; Junsei, Japan), absorbance was measured at 540 nm using an ELISAreader (Spectra MAX 250, Molecular Devices Co., USA). Each experimentwas repeated 3 times. The result is shown in FIG. 3.

As can be seen from FIG. 3, syringaresinol recovered cellular activity,which had been decreased due to smoking, to a level substantiallycomparable to the normal level. Accordingly, it can be seen thatsyringaresinol can detoxify smoking-induced toxicity by recoveringcellular activity which has declined due to smoking.

[Test Example 3] Evaluation of SIRT1 Expression Promoting Effect inAdipocytes, Hepatocytes and Myocytes

The SIRT1 gene expression promoting effect of syringaresinol in humanadipocytes, hepatocytes and myocytes was evaluated as follows.

Human adipocytes, hepatocytes and myocytes were purchased from Zen-Bio(Research Triangle Park, N.C., USA) and cultured in a 5% CO₂ incubatorusing respectively an adipocyte medium (OM-AM, Zen-Bio), a hepatocytemedium (HM-2, Zen-Bio) and a myocyte medium (SKM-D, SKM-M, Zen-Bio). Thecells were treated with syringaresinol dissolved in DMSO to aconcentration of 20, 50 or 100 μM for 24 hours. A negative control groupwas treated with DMSO corresponding to 1/1000 of the volume of theculture medium.

The cells treated with each sample were washed 2 times with cold PBS andRNA was extracted therefrom using the TRIzol reagent (Invitrogen). Then,cDNA was synthesized from the extracted RNA (5 mg) using a reversetranscription system (Fermentas, Glen Burnie, Md., USA). The expressionpattern of the SIRT1 and GAPDH genes was monitored by qRT-PCR using thesynthesized cDNA and predesigned primers and probes. PCR reaction andanalysis were carried out using the Rotor-Gene 3000 system (CorbettResearch, Sydney, Australia). The result is shown in FIG. 4.

As can be seen from FIG. 4, syringaresinol increased SIRT1 expression inhuman adipocytes, hepatocytes and myocytes in a concentration-dependentmanner. Accordingly, it can be seen that syringaresinol can improvemetabolic disorder, specifically lipid metabolic disorder.

[Test Example 4] Evaluation of Ability to Regulate Expression of FatMetabolism-Related Genes in Adipocytes and Hepatocytes

Human adipocytes, hepatocytes and myocytes were treated with 50 μMsyringaresinol and washed with PBS in substantially the same manner asin Test Example 3. RNA was extracted from the cells and cDNA wassynthesized therefrom. The change in expression pattern of fatty acidsynthesis-related genes such as ADD1/SREBP1c, ACC, FAS, etc. and fattyacid oxidation-related genes such as ACO, CPT1, mCAD, etc. was measuredby qRT-PCR. The result of comparing with a control group which wastreated only with DMSO is shown in FIGS. 5-10.

As can be seen from FIGS. 5-7, syringaresinol suppressed the expressionof fatty acid synthesis-inducing genes. Also, as can be seen from FIGS.8-10, syringaresinol increased the expression of fatty acid oxidationpromoting genes. Accordingly, it can be seen that syringaresinol canprevent fat accumulation by suppressing fat synthesis and at the sametime increasing fat consumption.

[Test Example 5] Evaluation of Fatty Acid Oxidation Promoting Ability

Human adipocytes, hepatocytes and myocytes were treated with 50 μMsyringaresinol in substantially the same manner as in Test Example 3.The cells were washed with PBS and cultured for a day in a medium forfatty acid oxidation analysis. The medium was recovered and the amountof ³H₂O was measured. A result of comparing with a non-treated group isshown in FIG. 11.

As can be seen from FIG. 11, syringaresinol increased fatty acidoxidation in human adipocytes, hepatocytes and myocytes. Accordingly, itcan be seen that syringaresinol can suppress fat accumulation in thebody.

[Test Example 6] Evaluation of PGC-1 Expression Promoting Effect ofSyringaresinol

Human adipocytes, hepatocytes and myocytes were treated with 50 μMsyringaresinol in substantially the same manner as in Test Example 3 andwashed with PBS. Then, RNA and cDNA were sequentially extractedtherefrom. The mRNA expression of the energy metabolism-related genesPGC-1α and PGC-1β was analyzed by qRT-PCR. A result of comparing theexpression of PGC-1α and PGC-1β with a control group treated only withDMSO is shown in FIG. 12 and FIG. 13, respectively.

As can be seen from FIG. 12 and FIG. 13, syringaresinol increased theexpression of PGC-1α and PGC-1β in human adipocytes, hepatocytes andmyocytes. Accordingly, it can be seen that syringaresinol can suppressfat accumulation in the body by facilitating energy metabolism.

[Test Example 7] Evaluation of SIRT1 Expression Promoting Effect in AgedHuman Retinal Epithelial Cells

The SIRT1 gene expression promoting effect of syringaresinol in agedhuman retinal epithelial cells was evaluated as follows.

Human retinal endothelial cell line ARPE-19 was purchased from ATCC(Manassa, Va., USA) and cultured in a 5% CO₂ incubator using DMEM (GibcoBRL, Grand Island, N.Y., USA) containing 10% bovine serum, 1%penicillin/streptomycin, amphotericin B and an antifungal agent until70% confluency. The aging of the retinal cells was induced bysubculturing until they did not grow any more. The population doublinglevel (PDL) was calculated according to the following equation for eachgeneration until the cell growth was stopped. The PDL value is higher inaged cells.PDL=(Log₁₀ Y−Log₁₀ X)/Log₁₀2

Y: number of cells at the end of the generation

X: number of cells at the beginning of the generation

5 PDL cells were treated with syringaresinol dissolved in DMSO at aconcentration of 50 or 100 μM every other day while inducing aging to 15PDL cells. The cells of a negative control were treated with DMSOcorresponding to 1/1000 of the volume of the culture medium.

The cells treated with each sample were washed 2 times with cold PBS andprotein was isolated using the RIPA buffer (Santa Cruz Biotechnology,Santa Cruz, Calif., USA). The isolated protein was quantitated using theBCA assay kit (Pierce, Rockford, USA) and 30 μg was subjected to westernblotting using the iBlot Dry blotting system (Invitrogen, Carlsbad,Calif., USA). The SIRT1 protein expression level was measured usinganti-SIRT1 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA).The result is shown in FIG. 14.

As can be seen from FIG. 14, syringaresinol increased SIRT1 expression,which had been decreased in aged retinal cells, in aconcentration-dependent manner. In particular, 100 μM syringaresinolincreased SIRT1 expression to a level comparable to that of youngretinal epithelial cells. Accordingly, it can be seen thatsyringaresinol can prevent or improve disease, particularly age-relatedeye disease, by increasing SIRT1 expression in retinal cells.

[Test Example 8] Evaluation of Mitochondrial Biosynthesis PromotingEffect in Aged Retinal Cells

The aging of retinal cells was induced while treating with 50 or 100 μMsyringaresinol or with DMSO as a negative control, in substantially thesame manner as in Test Example 7. The cells treated with each samplewere washed 2 times with cold PBS and genomic DNA (gDNA) was extractedtherefrom using the FastPure DNA kit (Tokyo, Japan). For evaluation ofmitochondrial biosynthesis, the quantity of the mitochondrial DNA markercytochrome oxidase subunit II and the nuclear DNA marker cyclophilin Awas quantified through real-time PCR using the extracted gDNA and thenumber of mitochondria was measured by calculating the quantity ofmitochondrial DNA relative to the nuclear DNA. PCR reaction and analysiswere carried out using the Rotor-Gene 3000 system (Corbett Research,Sydney, Australia). The result is shown in FIG. 15.

As can be seen from FIG. 15, the cells treated with 50 and 100 μMsyringaresinol exhibited about 40% and 60% increased DMSO. Accordingly,it can be seen that syringaresinol can prevent or improve eye disease byincreasing mitochondrial biosynthesis in aged retinal cells in aconcentration-dependent manner.

[Test Example 9] Evaluation of Mitochondrial Function Recovering Effectin Aged Retinal Cells

The aging of retinal cells was induced while treating with 50 or 100 μMsyringaresinol or with DMSO as a negative control, in substantially thesame manner as in Test Example 7. For evaluation of mitochondrialfunction recovering effect, the level of energy (ATP) and reactiveoxygen species production in retinal cells was investigated. The energyproduction was determined by recovering the aged cells after washingwith PBS and then with hot water and measuring luminescence using theATP determination kit (Molecular Probes, Eugene, Oreg., USA) and theTecan system (Infinite M200, Tecan, Austria). The reactive oxygenspecies production was measured using a flow cytometer (BD Biosciences,San Jose, Calif., USA) after washing the aged cells with PBS andstaining them with the reactive oxygen species detection reagent(H₂DCFDA, Invitrogen, Carlsbad, Calif., USA). The result is shown inFIG. 16.

As can be seen from FIG. 16, the cells treated with 50 and 100 μMsyringaresinol exhibited about 21% and 33% increased energy (ATP)production and about 7% and 11% decreased reactive oxygen speciesproduction (significant), as compared to the cells treated only withDMSO. Accordingly, it can be seen that syringaresinol can prevent orimprove eye disease by increasing energy production and at the same timedecreasing reactive oxygen species production in retinal cells in aconcentration-dependent manner.

[Test Example 10] Evaluation of SIRT1 Expression Promoting Effect inHuman PBMCs

The SIRT1 gene expression promoting effect of syringaresinol in humanPBMCs was evaluated as follows.

Human PBMCs were purchased from Zen-Bio (Research Triangle Park, N.C.,USA) and cultured in a 5% CO₂ incubator using a PBMC medium (Zen-Bio)until 80% confluency. Then, the cells were treated for 24 hours withsyringaresinol dissolved in DMSO to a concentration of 20, 50 or 100 μM.A negative control group was treated with DMSO corresponding to 1/1000of the volume of the culture medium. The cells treated with each samplewere washed with PBS and RNA was extracted therefrom using the TRIzolreagent (Invitrogen). Then, cDNA was synthesized from the extracted RNA(5 μg) using a reverse transcription system (Fermentas, Glen Burnie,Md., USA). The expression pattern of the SIRT1 and GAPDH genes wasmonitored by qRT-PCR using the synthesized cDNA and predesigned primersand probes. qRT-PCR reaction and analysis were carried out using theRotor-Gene 3000 system (Corbett Research, Sydney, Australia). The resultis shown in FIG. 17.

As can be seen from FIG. 17, syringaresinol increased SIRT1 expressionin PBMCs in a concentration-dependent manner. Accordingly, it can beseen that syringaresinol can enhance immunity and prevent and improveimmune disease by increasing SIRT1 expression in peripheral bloodmononuclear cells in a concentration-dependent manner.

[Test Example 11] Evaluation of Reactive Oxygen Species ProductionInhibiting Effect in Human PBMCs

After pretreating human PBMCs with 20, 50 or 100 μM syringaresinol insubstantially the same manner as in Test Example 10, followed by washingwith PBS, inflammatory response was induced by treating with 10 ng/mLlipopolysaccharide (LPS). After treating the cells with H₂-DCFDA(Invitrogen), which is a fluorescent substance capable of detectingreactive oxygen species, the amount of fluorescent substance wasmeasured using a multiplate reader (Infinite M200; Tecan, Mannedorf,Switzerland). The result is shown in FIG. 18.

As can be seen from FIG. 18, the cells treated with syringaresinolexhibited about 60% decreased reactive oxygen species production ascompared to the cells treated only with LPS. Accordingly, it can be seenthat syringaresinol can enhance immunity and prevent and improve immunedisease by reducing reactive oxygen species and thereby suppressinginflammatory response.

[Test Example 12] Evaluation of Effect of Inhibiting Expression ofInflammation-Related Genes in Human PBMCs

After pretreating human PBMCs with 50 μM syringaresinol, inflammatoryresponse was induced by treating with 10 ng/mL LPS, in substantially thesame manner as in Test Example 11. The cells treated with each samplewere washed with PBS and RNA and cDNA were extracted and synthesizedtherefrom. Then, the expression pattern of the inflammatoryresponse-related genes IL-1b, IL-6. iNOS, COX2, MMP9 and CCR2 wasmonitored using the synthesized cDNA and predesigned primers. The resultis shown in FIG. 19.

As can be seen from FIG. 19, the cells treated with syringaresinolexhibited about 90% decreased expression of the inflammatoryresponse-related genes as compared to the cells treated only with LPS.Accordingly, it can be seen that syringaresinol can enhance immunity andprevent or improve immune disease by reducing inflammatory response.

[Test Example 13] Evaluation of Human PBMC Migration and DepositionInhibiting Effect

After treating human PBMCs with 20, 50 or 100 μM syringaresinol or DMSOin substantially the same manner as in Test Example 11, inflammatoryresponse was induced by treating with 10 ng/mL TNF. After treatingdifferentiated 3T3-L1 adipocytes (Zen-Bio) with the PBMCs, it wasinvestigated whether the PBMCs migrate onto the adipocytes and deposit.48 hours later, after removing the PBMCs remaining without migrating,the cells were detached from a culture dish using trypsin/EDTA and thencounted using a cell counter. The result is shown in FIG. 20.

As can be seen from FIG. 20, syringaresinol inhibited the migration anddeposition of the PBMCs onto the adipocytes by about 50%. Since thissuggests that syringaresinol can inhibit the migration and deposition ofperipheral blood mononuclear cells in response to inflammatory signals,it can be seen that syringaresinol can enhance immunity and prevent orimprove immune disease by suppressing inflammatory response.

[Test Example 14] Evaluation of Effect of Inducing Change in Human PBMCType

After treating human PBMCs with 20, 50 or 100 μM syringaresinol or DMSOin substantially the same manner as in Test Example 11, RNA wasextracted from the PBMCs and cDNA was synthesized therefrom in order tomeasure the expression of type 2 immune cell-related genes. Theexpression pattern of the transcriptional regulators necessary forinducement of differentiation to type 2 immune cells, such as PGC-1α andPGC-1β, and the genes mainly expressed in type 2 immune cells, such asIL-10 and arginase I, was measured using the synthesized cDNA. Theresult is shown in FIG. 21.

As can be seen from FIG. 21, the cells treated with syringaresinolexhibited significantly increased expression of type 2 immunecell-specific marker genes. Accordingly, it can be seen thatsyringaresinol can enhance immunity and prevent or improve immunedisease by promoting differentiation of peripheral blood mononuclearcells into type 2 immune cells.

Hereinafter, the present disclosure will be described in detail throughformulation examples. However, the formulation examples are forillustrative purposes only and it will be apparent to those of ordinaryskill in the art that the scope of the present disclosure is not limitedby the formulation examples.

[Formulation Example 1] Health Food

Syringaresinol 1000 mg Vitamin mixture Vitamin A acetate 70 μg Vitamin E1.0 mg Vitamin B₁ 0.13 mg Vitamin B₂ 0.15 mg Vitamin B₆ 0.5 mg VitaminB₁₂ 0.2 μg Vitamin C 10 mg Biotin 10 μg Nicotinamide 1.7 mg Folic acid50 μg Calcium pantothenate 0.5 mg Mineral mixture Ferrous sulfate 1.75mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassium dihydrogenphosphate 15 mg Calcium monohydrogen phosphate 55 mg Potassium citrate90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

Although the above-described mixing ratios of the vitamin and mineralmixtures are provided as specific examples suitable for health food, themixing ratios may be changed as desired.

[Formulation Example 2] Health Drink

Syringaresinol 1000 mg Citric acid 1000 mg Oligosaccharide 100 g Taurine1 g Purified water balance

According to a commonly employed method, the above-described ingredientsare mixed and stirred for about 1 hour while heating at about 85° C. Theresulting solution is filtered and sterilized.

[Formulation Example 3] Tablet

Granules formed by mixing 100 mg of syringaresinol g, 50 mg of soybeanextract, 100 mg of glucose, 50 mg of red ginseng extract, 96 mg ofstarch and 4 mg of magnesium stearate and adding 40 mg of 30% ethanolare dried at 60° C. and prepared into a tablet.

[Formulation Example 4] Granule

Granules formed by mixing 100 mg of syringaresinol g, 50 mg of soybeanextract, 100 mg of glucose and 600 mg of starch and adding 100 mg of 30%ethanol are dried at 60° C. and filled in a pouch.

[Formulation Example 5] Ointment

An ointment is prepared according to a commonly employed method with thecomposition described in Table 1.

TABLE 1 Ingredients Content (wt %) Syringaresinol 3.0 Glycerin 8.0Butylene glycol 4.0 Liquid paraffin 15.0 β-Glucan 7.0 Carbomer 0.1Caprylic/capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Beeswax 4.0 Preservative,pigment and fragrance adequate Purified water balance

INDUSTRIAL APPLICABILITY

A composition according to the present disclosure, which contains anSIRT1 activating agent, has superior detoxifying effect, particularlysuperior effect of detoxifying smoking-induced toxicity.

The composition according to the present disclosure sure, which containsan SIRT1 activating agent, has excellent effect of preventing andimproving metabolic disorder, specifically obesity, type 2 diabetes,hyperlipidemia or fatty liver.

The composition according to the present disclosure sure, which containsan SIRT1 activating agent, has excellent effect of preventing orimproving eye disease, particularly age-related eye disease.

The composition according to the present disclosure sure, which containsan SIRT1 activating agent, has excellent effect of preventing orimproving immune disease such as allergy, atopic dermatitis, hay feveror rheumatoid arthritis.

The invention claimed is:
 1. A method for treating immune disease in asubject suffering from immune disease comprising administering aneffective amount of syringaresinol or a pharmaceutically acceptable saltthereof to the subject wherein the syringaresinol treats immune diseasein the subject, and wherein the immune disease is selected from thegroup consisting of allergy, atopic dermatitis, hay fever, andrheumatoid arthritis.
 2. The method according to claim 1, wherein thesyringaresinol is administered in a form of agent, and wherein the agentcomprises syringaresinol in an amount of 0.0001-10 wt % based on thetotal weight of the agent.
 3. The method according to claim 1, whereinthe syringaresinol is included in an extract of one or more selectedfrom flax seed, phellodendri cortex, acanthopanacis cortex, sesame seedand ginseng berry.
 4. The method according to claim 1, wherein themethod is for promoting SIRT1 expression and conversing to type 2 immunecells in peripheral blood mononuclear cells in the subject by activatingSIRT1.
 5. The method according to claim 2, wherein the agent isadministered in a form of a composition, and the composition comprises0.001-20 wt % of the agent based on the total weight of the composition.6. The method according to claim 5, wherein the composition is a foodcomposition.
 7. The method according to claim 5, wherein the compositionis a pharmaceutical composition.